The long-term objective of this research is to understand the molecular basis of the physiological functions of inwardly rectifying K+ (IRK) channels. IRK) channels, IRK channels are abundant in brain, heart, kidney, and pancreas. They are highly selective for K+ ions and conduct more K+ into cells than out of them, a property called inward rectification that underlies their crucial role in maintaining the resting membrane potential and regulating neuronal excitability, heart beat, and hormone secretion. Malfunction of IRK channels is known to cause neurological disorders in animal models. Thus, elucidation of the molecular mechanisms of ion permeation and inward rectification is important for understanding the physiological functions of inwardly rectifying K+ channels under normal conditions and in disorders such as epilepsy, arrhythmia, and diabetes. This research project focuses on a strongly rectifying channel, IRK1, that is abundant in both brain and heart. We propose to investigate the structural features of the ion permeation pathway, or pore, and the molecular determinants of K+ selectivity and inward rectification. Genetically altered IRK1 channels will be expressed in Xenopus frog oocytes and studied electrophysiologically using two-electrode voltage- clamp and patch-clamp. The specific aims are: (1) to map the structural domains involved in forming the internal portion of the channel pore, where cytoplasmic cations such as Mg2+ and polyamines bind to produce inward rectification; and to identify amino acid residues lining the ion conduction pathway in the H5 pore loop, which most likely forms the K+ selectivity filter and external entryway of the channel pore; (2) to study the molecular mechanism of K+ selectivity using the unnatural amino acid mutagenesis method: and (3) to localize amino acid residues in the inner pore that contribute to Mg2+ and polyamine binding; and to study the biophysical mechanism of interactions between permeant K+ ions and the blockers. Results from this research will help us understand better in general how inwardly rectifying K+ channels function. They will also allow us to gain better insight into the molecular architecture of the channel pore, which may help in the development of pharmacological agents directed at these channels.